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For Research Use Only. Not for use in diagnostic procedures.
Eureka™ Genotyping Workflow User Guide
Information in this document is subject to change without notice.
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P/N 703400
2 Eureka™ Genotyping Workflow User Guide
Contents
Chapter 1 Introduction ...................................................................................................... 5
About the Eureka™ Genotyping Assay ................................................................................... 5
Overview of the Eureka™ Genotyping Assay ......................................................................... 6
Related Documentation ........................................................................................................... 8
Chapter 2 Safety ................................................................................................................ 9
Warnings and Precautions ...................................................................................................... 9
Special Requirements .............................................................................................................. 9
VIAFLO 384 Instrument ........................................................................................................ 10
Chapter 3 Genomic DNA General Requirements .......................................................... 11
Chapter 4 Preparation Before Starting ............................................................................ 12
Thermal Cycler ..................................................................................................................... 12
Thermal Cycler Programs ................................................................................................................................ 12
Working with Plates .............................................................................................................. 14
Before Using the INTEGRA VIAFLO 384 Instrument ............................................................ 15
Two-step Pipette Tip Loading Method .................................................................................. 16
Testing the Heated Plate Sealer ............................................................................................. 20
Equipment, Consumables, and Reagents Required ............................................................... 21
Reagents ............................................................................................................................................................. 21
Pre-PCR Clean Lab ........................................................................................................................................... 22
Reagents Required in Pre-PCR Clean Lab..................................................................................................... 22
Equipment Required in Pre-PCR Clean Lab ................................................................................................. 22
Labware Required in Pre-PCR Clean Lab ..................................................................................................... 23
Post-PCR Lab ........................................................................................................................ 25
Reagents Required in Post-PCR Lab .............................................................................................................. 25
Equipment Required in Post-PCR Lab .......................................................................................................... 26
Labware Required in Post-PCR Lab ............................................................................................................... 27
Preparation of DNA Wash Buffer .................................................................................................................. 29
VIAFLO 384 Programs using a 12.5 μL Head ........................................................................ 29
Chapter 5 Fill 384 Plates with Eureka™ Genotyping Assay Reagents ........................... 30
Load Tips for Plate Filling Operations ................................................................................... 30
Chapter 6 Eureka™ Genotyping Assay Protocol ............................................................ 31
Eureka™ Genotyping Workflow User Guide 3
Preparing the Genomic DNA 384 Plate ................................................................................. 31
Stage 1: DNA Heat Fragmentation ........................................................................................ 32
Duration ............................................................................................................................................................. 32
Materials Required ........................................................................................................................................... 32
Procedure ........................................................................................................................................................... 32
Dispense the Eureka™ Genotyping Panel (Just Prior to Use) ................................................ 33
Duration ............................................................................................................................................................. 33
Reagents and Materials Required ................................................................................................................... 33
Procedure ........................................................................................................................................................... 34
Stage 2: Hybridization with Eureka™ Genotyping Panel ...................................................... 35
Duration ............................................................................................................................................................. 35
Reagents and Materials Required ................................................................................................................... 35
Procedure ........................................................................................................................................................... 35
Dispense the Eureka™ Ligation Solution .............................................................................. 37
Duration ............................................................................................................................................................. 37
Reagents and Materials Required ................................................................................................................... 37
Procedure ........................................................................................................................................................... 38
Stage 3: Ligation .................................................................................................................... 39
Duration ............................................................................................................................................................. 39
Reagents and Materials Required ................................................................................................................... 39
Procedure ........................................................................................................................................................... 40
Prepare PCR Sample Index Plate ........................................................................................... 41
Duration ............................................................................................................................................................. 42
Reagents and Materials Required ................................................................................................................... 42
Procedure ........................................................................................................................................................... 43
Stage 4: PCR Amplification ................................................................................................... 46
Duration ............................................................................................................................................................. 46
Reagents and Materials Required ................................................................................................................... 46
Procedure ........................................................................................................................................................... 46
Chapter 7 Post-PCR Library Pooling .............................................................................. 48
Stage 1: Library Pooling ........................................................................................................ 48
Materials Required ........................................................................................................................................... 48
Pooling the Library ........................................................................................................................................... 48
4 Eureka™ Genotyping Workflow User Guide
DNA Wash Buffer ............................................................................................................................................. 49
Stage 2: Pooled Library Clean-up and Concentration ............................................................ 50
Reagents and Materials Required ................................................................................................................... 50
Stage 3: Library Qualification and Quantification via Agilent 2100 Bioanalyzer .................... 52
Reagents and Materials Required ................................................................................................................... 52
Stage 4: Combining Pooled Libraries ..................................................................................... 54
Library Blending and Balancing ..................................................................................................................... 54
Chapter 8 Sequence Data Generation ............................................................................. 55
Chapter 9 Processing Multiple Plates per Week in the Eureka™ Genotyping Workflow ......................................................................................................................................... 57
Overview of Eureka™ Genotyping Workflows ..................................................................... 57
Assumptions ...................................................................................................................................................... 57
Appendix A Eureka™ Genotyping Workflow Assay FAQs and Troubleshooting ....... 59
FAQs ..................................................................................................................................... 59
Troubleshooting .................................................................................................................... 60
Eureka™ Genotyping Workflow User Guide 5
Chapter 1 Introduction
About the Eureka™ Genotyping Assay
Eureka™ Genotyping Solution introduces a new genotyping technology platform that utilizes next
generation sequencing to enable genotyping of hundreds to thousands of DNA samples for tens to
thousands of loci. It can be used to interrogate SNPs, and small to large insertions/deletions (indels).
The Eureka Genotyping Assay is a ligation-dependent PCR reaction, which uses interrogation site barcodes
contained within the hybridization probes, as well as sample index barcodes added during the
amplification step. Starting with genomic DNA, the samples are processed in a 384 plate format to generate
next generation sequencing libraries for loading onto a sequencer. Short cycle sequence data is generated
from the prepared libraries and Eureka™ Analysis Suite (EAS) software is used to assign each read to a
sample, locus, and allele combination. From statistical analysis of the tabulated reads, the EAS software
infers the genotype of each sample for each locus.
6 Eureka™ Genotyping Workflow User Guide
Overview of the Eureka™ Genotyping Assay
The Eureka Genotyping Assay involves the following steps:
1. Isolating/Preparing Genomic DNA—resulting in samples that meet requirements indicated in Chapter
3, Genomic DNA General Requirements.
2. Creating a Eureka Genotyping library—resulting in next generation sequencing (NGS) ready
library, as depicted in Figure 1.1.
See Chapter 6, Eureka™ Genotyping Assay Protocol and Chapter 7, Post-PCR Library Pooling.
3. NGS data generation. See Chapter 8, Sequence Data Generation.
4. Sequence data analysis.
A list of the required equipment and supplies for running the Eureka Genotyping Assay can be found in
the Eureka™ Genotyping Workflow Site Preparation Guide, P/N 703401, or in the section Equipment,
Consumables, and Reagents Required.
Figure 1.1 Illustration of the Eureka™ Genotyping Assay, outlining the various steps of the assay
workflow.
A set of three probes is used to interrogate each marker of interest. Each “probe triplet” targets one
marker/variant. The probes are designed with a hybridization region targeting the genomic DNA
and a common PCR priming region. Hundreds to thousands of probe triplets are in a single
Eureka™ Genotyping Panel.
The sample DNA and Eureka Genotyping Panel are mixed and hybridized.
The two left probes have the same hybridization sequence other than the last base and they
compete to match with the base of interest. The right probe hybridizes to the target DNA sequence
3' to the base of interest.
A ligase is added to join adjacent left and right probes.
Eureka™ Genotyping Workflow User Guide 7
PCR amplification proceeds from primers that target the common PCR primer sites found on
ligated probes and also contain a unique sample index sequence that is used to associate the allele x
locus barcode sequence with a sample. The PCR primers also contain sequences required for NGS
data generation.
Each usable sequence read set contains an allele and locus barcode and a sample index. Based on
the barcode and sample index, the number of reads assigned to each sample, locus, and allele
combination are tabulated.
The overall workflow for Eureka Genotyping Solution is laid out in Figure 1.2 below.
Figure 1.2 An overview of the Eureka™ Genotyping Solution workflow.
In summary, the Eureka Genotyping Solution is a product line that provides catalog and custom
genotyping panels that can be used to generate genotype information on thousands of samples per week. It
enables economical low- and mid-plex genotyping that is both fast and flexible.
8 Eureka™ Genotyping Workflow User Guide
Related Documentation
Eureka™ Genotyping Workflow Site Preparation Guide, P/N 703401
Eureka™ Genotyping Workflow Quick Reference Card, P/N 703402
Eureka™ Analysis Suite User Guide, P/N 703380
Eureka™ Genotyping Workflow User Guide 9
Chapter 2 Safety
Warnings and Precautions
Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or
animals. Do not use internally or externally in humans or animals. Copies of the Safety Data Sheets for the
kit components are available on our web site.
Caution: All chemicals should be considered potentially hazardous. Therefore, we recommend that this
product should be handled only by individuals who have been trained in laboratory techniques and used
in accordance with the principles of good laboratory practice. Wear suitable protective clothing, such as
gloves, a lab coat, and safety glasses. Care should be taken to avoid contact with skin and eyes. In case of
contact with skin or eyes, wash immediately with water. See Safety Data Sheet (SDS) for specific advice.
WARNING: Under certain pressure conditions and heated lid failure, reaction that has condensed onto the foil seal can spray out of the wells when the foil seal is removed.
WARNING: Do not close the heated plate sealer with the block inside.
Special Requirements
Precautions are required when manipulating genomic DNA to avoid contamination with DNA amplified in
other reactions and procedures. These precautions minimize possible sources of contamination that could
reduce genotyping accuracy, call rate, and consequently, genetic power. To reduce the possibility of cross-
contamination, we recommend maintaining a single direction workflow from a Pre-PCR Lab to a Post-PCR
Lab.
NOTE:
The most likely source of contamination for the Eureka Genotyping Assay is previously
amplified PCR product.
Each room should contain dedicated equipment such as microfuges, pipettes, tips, etc.
Once you enter the Post-PCR Lab, do not return to the Pre-PCR Clean Lab that day.
Maintain an ambient laboratory environment throughout the procedure.
Precautions to minimize contaminating pre-PCR steps with amplified PCR product include the following:
Store reagents in the proper room according to the box label and reagent kit insert.
Restrict movement through labs containing amplified DNA.
Use proper gowning procedures.
Print separate copies of the protocol for each room.
10 Eureka™ Genotyping Workflow User Guide
Chapter 2 | Safety 9
VIAFLO 384 Instrument
Use Figure 2.1 to become familiar with the VIAFLO 384 Instrument and the locations of the various
components and controls. For further information, please refer to the VIAFLO 96/384 Operating
Instructions (INTEGRA P/N 125950).
TIP: The Twin.Tec PCR plate 384 is ALWAYS on a VIAFLO PCR 384 Cooling Plate when on the VIAFLO 384 Instrument.
Figure 2.1 INTEGRA VIAFLO 384 Instrument
Eureka™ Genotyping Workflow User Guide 11
Chapter 3 Genomic DNA General Requirements
DNA MUST be dissolved in TE buffer at pH 8.3 (10 mM Tris HCl, 1 mM EDTA). DNA dissolved in
water cannot be used.
We recommend that the DNA concentration be 42 ng/μL (≈150 ng/reaction) as based on picogreen
measurements. Picogreen has a high specificity for dsDNA, and will not measure ssDNA, salts, or
enzyme inhibitors.
DNA should be free of enzyme inhibitors. Examples of inhibitors include high concentrations of
heme (from blood) and high concentrations of chelating agents (i.e., EDTA).
The DNA extraction/purification method should render DNA that is generally salt-free. High
concentrations of particular salts will affect hybridization and possibly downstream steps.
12 Eureka™ Genotyping Workflow User Guide
Chapter 4 Preparation Before Starting
This section provides information on procedures used each time the assay is performed and details on steps
that are critical to the success of the assay. It is essential that you are familiar with the information in this
section prior to running the Eureka™ Genotyping Assay. A list of all equipment and resources required is
in the Eureka™ Genotyping Workflow Site Preparation Guide, P/N 703401.
Room Temperature
When referred to in the Eureka Genotyping Assay, room temperature is 18°C to 25°C.
Control Recommendations
We recommend that the supplied control DNA be used in one well of every 96 well plate. There should be
4 control wells on each 384 plate of DNA. The location of the control wells needs to be correctly indicated in
the Sample to Well Mapping files as described in the Eureka™ Analysis Suite User Guide (P/N 703380).
TIP: If the locations of the control DNA are varied on each plate, they serve as plate tracking controls that are useful when processing multiple plates or to identify a plate that was not processed in the proper orientation.
Thermal Cycler
The performance of this assay has been verified on the following thermal cycler:
Bio-Rad Laboratories C1000 Touch™
The performance of this assay has not been verified with other thermal cyclers.
WARNING: Use of other thermal cyclers may result in assay failure and may violate the Eureka Genotyping Panel and Reagent replacement policy.
Thermal Cycler Programs
The thermal cycler programs listed below are used during this protocol. Before beginning the processing of
samples, enter and store these programs on the thermal cycler(s).
IMPORTANT: Use heated lid for all thermal cycler programs.
EG_Heat_Frag
Table 4.1 EG_Heat_Frag Thermal Cycler Program
Temperature Time
98°C 15 min
25°C Hold
Eureka™ Genotyping Workflow User Guide 13
EG_Hyb
Table 4.2 EG_Hyb Thermal Cycler Program
Temperature Time Cycles
98°C 1 min
60°C 20 hrs (may require hold
step or multiple
cycles to achieve 20
hrs total)
54°C 1 min
TIP: Many thermal cyclers can employ an indefinite hold step and be advanced manually from 60°C to 54°C after approximately 20 hours.
EG_Lig
Table 14.33 EG_Lig Thermal Cycler Program
Temperature Time
54°C 15 min
92°C 15 sec
8°C Hold
EG_PCR
Table 4.4 EG_PCR Thermal Cycler Program
Temperature Time Cycles
94°C 5 min 1
94°C 10 sec 32
65°C 30 sec
72°C 1 min 1
8°C Hold 1
14 Eureka™ Genotyping Workflow User Guide
Working with Plates
Labeling Plates
Position A1
It is good practice to label position A1 of all plates to avoid inverting a plate. Marking the A1 corner of
the plate with a lab marker is an easy way to aid visual confirmation of plate orientation.
Name Plates
Label the top and one side of all plates to avoid confusion when processing multiple plates.
Handling Plates
Maintaining Plate Orientation
It is critical to keep the plate orientation during the multiple sample transfer steps. Position A1 must be
in the same location for the source and destination plates. If one of the plates is in the wrong orientation
during a transfer step, samples will no longer correspond to their original plate locations.
Plate Removal From Thermal Cycler
Loosen the heated lid of the thermal cycler and open the lid slowly. This prevents the plates from
sticking to the lid of the thermal cycler.
Unsealing Plates
When removing plate seals, liquid MUST not splash out of the wells. It is helpful to have the
Twin.TecTM PCR Plate 384 in a metal block to help secure the plate while unsealing.
Seal and Spin
A protocol step to seal and spin a plate means:
Sealing the Plate
Refer to the temperature and sealing time that you have determined for your heated foil plate sealer
instrument. To find out the optimal temperature and sealing time, refer to Testing the Heated Plate Sealer.
Spin
Spin the plate at 800 xg (2000 rpm in a benchtop centrifuge with plate rotor) for 1 minute at room
temperature. The rpm values listed are only approximate. Please set your centrifuge as needed to
achieve the listed xg values.
Eureka™ Genotyping Workflow User Guide 15
Before Using the INTEGRA VIAFLO 384 Instrument
Water Run
Perform a water run with no DNA (using water instead of reagents) on the VIAFLO 384 prior to
running DNA samples to check protocols.
Pre-cool the VIAFLO PCR 384 Cooling Plates
When using the VIAFLO 384, a Twin.Tec PCR 384 plate is ALWAYS on a VIAFLO PCR 384 Cooling
Plate. The VIAFLO PCR 384 Cooling Plate is cold during Stage 3: Ligation and Stage 4: PCR Amplification.
The VIAFLO PCR 384 Cooling Plate (INTEGRA Biosciences, P/N 6255) can be pre-chilled on ice or
stored in a refrigerator (4°C) prior to use.
IMPORTANT: Do not store the VIAFLO PCR 384 Cooling Plate at –20°C as this results in freezing of the solutions in the Twin.Tec PCR Plate 384 when placed on the VIAFLO PCR 384 Cooling Plate.
The target temperature of the cooling plate for use in steps requiring cold temperatures is 2-4°C. The
VIAFLO PCR 384 Cooling Plates maintain a cool temperature on the VIAFLO instrument for
approximately 15 minutes.
IMPORTANT: Use only the VIAFLO PCR 384 Cooling Plates on the VIAFLO 384 instrument due to height and positional constraints of the VIAFLO 384 programs. Always be sure that the Twin.Tec PCR Plates are fully seated in the VIAFLO PCR 384 Cooling Plates with no bowing or warping of the PCR plates. Loose or bowed plates result in incorrect alignment, poor liquid transfer, and possible assay failure.
16 Eureka™ Genotyping Workflow User Guide
Two-step Pipette Tip Loading Method
The two-step pipette tip loading method ensures that the pipette tips are properly seated onto the VIAFLO
384 instrument head. It adds robustness to each transfer step of the protocol.
TIP: Make sure the VIAFLO 384 has one of the “EG” programs open to assist in loading of the tips.
1. Load 12 columns of tips onto the left half of VIAFLO 384 head:
1. Place an entire box of 12.5 μL filtered GripTips on Position A of the VIAFLO 384 instrument as
shown in Figure 4.1.
Figure 4.1 Step 1A: Two-step Pipette Tip Loading Method; Loading Tips onto Left Half of the VIAFLO 384
Head
2. Align the pipetting unit such that the left half of the columns on the VIAFLO 384 head are in
position with the right half of the tips in the tip box (12 columns of tips) as shown in Figure 4.2.
Figure 4.2 Step 1B: Two-step Pipette Tip Loading Method; Loading Tips onto Left Half of the VIAFLO 384
Head
Eureka™ Genotyping Workflow User Guide 17
3. Lower the pipetting unit until the tip load button on the top of the instrument flashes.
4. Load half the tips onto the VIAFLO 384 head.
Figure 4.3 Step 1D: One half box of tips (12 columns) loaded onto the left half of the VIAFLO 384 head.
2. Load 12 columns of tips onto the right half of VIAFLO 384 head:
1. Rotate the tip box so that the remaining tips are positioned on the right side of the box, in Position
A of the VIAFLO 384 instrument (Figure 4.4-A).
2. Lower the pipetting unit slowly until the remaining tips are in position with remaining columns of
the VIAFLO 384 head. The previously loaded tips go into the empty wells in the tip box (Figure 4.4-
B).
Figure 4.4 Steps 2A and 2B: Two-step Pipette Tip Loading Method; Loading Tips onto Right Half of
the VIAFLO 384 Head
3. Load the other half of the tip on the VIAFLO 384 head when the tip load button on the top of the
instrument flashes (Figure 4.5-A).
4. Ensure that all the tips are properly loaded by visually inspecting the head to see if the tips are
fully seated and flush with the head plate (Figure 4.5-B). Refer to Figure 4.6 for examples of loaded
tips. If tips appear to have improperly loaded, please return the tips to the tip box and repeat the
Two-step Pipette Tip Loading Method.
18 Eureka™ Genotyping Workflow User Guide
Figure 4.5 Steps 2C and 2D: Two-step Pipette Tip Loading Method; Loading Tips onto Right Half of the
VIAFLO 384 Head
Figure 4.6 Examples of Tip Attachments
Eureka™ Genotyping Workflow User Guide 19
20 Eureka™ Genotyping Workflow User Guide
Testing the Heated Plate Sealer
Sealers vary in both time and temperature settings that yield optimal results. Determine the time needed
for your heated plate sealer by following these steps:
1. For each time or temperature setting to be tested, heat seal an empty Twin.Tec PCR Plate 384 and
inspect the uniformity of the seal across the wells. Peel off the first seal and discard the seal (keep the
empty plate). Fill the wells of the previously sealed plate with 8 μL of water per well, reseal the plate
with a new, unused, foil seal. Weigh the plate, and incubate in a thermal cycler for 20 hr at 60°C.
2. After the 20 hr incubation visually inspect the wells and re-weigh the plate.
3. Weight loss of 0.2g or greater indicates improper sealing.
4. Unseal the plate and inspect the well openings. Highly deformed well openings indicates over-sealing.
IMPORTANT: Always ensure that plates are tightly sealed. An optimized seal prevents sample loss, cross-well contamination and well deformation. Over-sealing causes excessive deformation and potential cross contamination due to difficulties unsealing the plate. Under-sealing leads to evaporation and reaction failure.
Figure 4.7 Well deformation from two sealing times.
Figure 4.7 shows the results from two plate sealing protocols. In this instrument, both protocols are
sufficient for minimal evaporation. However, the five second protocol (right) causes oversealing. We
recommend retesting each heated plate sealer at least once a year to ensure proper sealing of the plates.
Eureka™ Genotyping Workflow User Guide 21
Equipment, Consumables, and Reagents Required
The equipment and consumables listed in this section have been tested and validated for use in this assay.
We do not recommend substituting either equipment, consumables, or reagents without consulting Thermo
Fisher Scientific. Substitutions are likely to compromise the assay results.
Reagents
Store all kit components at –20°C and avoid freeze-thaw.
Table 4.5 Reagents
Reagent Supplier Part Number Quantity
Eureka™ Genotyping Panel (2.5 mL) Thermo Fisher Custom P/N 1 vial
Eureka™ Control gDNA (20 μL) Thermo Fisher Species-
specific 1 vial
Eureka™ Ligation Solution (5.5 mL) Thermo Fisher 19551 1 vial
Eureka™ PCR Master Mix (3.5 mL) Thermo Fisher 19552 1 vial
Eureka™ Sample Index Plate (one of the
following):
Thermo Fisher
Eureka™ Sample Index Plate A, Indices 1–384
19702 Plate of 384
indices
Eureka™ Sample Index Plate B, Indices 385–768
19703 Plate of 384
indices
Eureka™ Sample Index Plate C, Indices 769–1152
19704 Plate of 384
indices
Eureka™ Sample Index Plate D, Indices 1153–
1536 19705 Plate of 384
indices
Eureka™ Sample Index Plate E, Indices 1537–
1920 19706 Plate of 384
indices
Eureka™ Sample Index Plate F, Indices 1921–
2304 19707 Plate of 384
indices
Eureka™ Sample Index Plate G, Indices 2305–
2688 19708 Plate of 384
indices
Eureka™ Sample Index Plate H, Indices 2689–
3072 19709 Plate of 384
indices
Eureka™ Sample Index Plate I, Indices 3073–3456
19710 Plate of 384
indices
Eureka™ Sample Index Plate J, Indices 3457–3840
19711 Plate of 384
indices
22 Eureka™ Genotyping Workflow User Guide
Pre-PCR Clean Lab
The Pre-PCR Clean Lab (or dedicated area such as a biosafety hood) should be free of PCR amplicons.
Reagents Required in Pre-PCR Clean Lab
Table 4.6 Reagents Required in Pre-PCR Clean Lab
Reagent Supplier Part Number
Eureka™ Genotyping Panel Kit Thermo Fisher Varies
Eureka™ Ligation Solution Thermo Fisher 19551
Eureka™ PCR Master Mix Thermo Fisher 19552
Eureka™ Sample Index Plates (as required) Thermo Fisher Varies
Equipment Required in Pre-PCR Clean Lab
Table 4.7 Equipment Required in Pre-PCR Clean Lab
Quantity Item Supplier Part Number
Minimum of 1 C1000 Touch™ Thermal Cycler with 384-well
block
Bio-Rad
Laboratories
1851138
1 VIAFLO 384 INTEGRA
Biosciences
6031
384-channel electronic pipette
1 384-channel pipetting head 0.5–12.5 μL 6131
1 Spring loaded plate holder A with slide function 6215
1 Spring loaded plate holder B with slide function 6220
Minimum of 2 VIAFLO PCR 384 Cooling Plate INTEGRA
Biosciences
6255
1 Centrifuge with mulitwell plate adaptor Various —
1 Bio-Rad PX™ 1 PCR Plate Sealer Bio-Rad
Laboratories
1814000
1 Pipette, single channel (10 μL) Various —
1 Vortex mixer with flat head Various —
Eureka™ Genotyping Workflow User Guide 23
Labware Required in Pre-PCR Clean Lab
Table 4.8 Labware Required in Pre-PCR Clean Lab
Labware Supplier & Part Number
Required for 384 Rxn
Image
Twin.TecTM PCR
Plate 384—blue
Part of the
Eureka™
Consumables
Kit P/N 902911
1 Plate
Twin.TecTM PCR
Plate 384—green
Part of the
Eureka™
Consumables
Kit P/N 902911
1 Plate
Twin.TecTM PCR
Plate 384—yellow
Part of the
Eureka™
Consumables
Kit P/N 902911
1 Plate
24 Eureka™ Genotyping Workflow User Guide
Labware Supplier & Part Number
Required for 384 Rxn
Image
Twin.TecTM PCR
Plate 384—red
Part of the
Eureka™
Consumables
Kit P/N 902911
1 Plate
12 column
reservoir, low
profile, divided
pyramid bottom,
84 mL (sterile)
Part of the
Eureka™
Consumables
Kit
P/N 902911
2 Reservoirs
Aluminum heat
seals
(pre-cut)
Part of the
Eureka™
Consumables
Kit
P/N 902911
5 Seals
12.5 μL filtered
GripTips
INTEGRA
Biosciences
P/N 6455 or
P/N 6459
4.25 Racks
Eureka™ Genotyping Workflow User Guide 25
Post-PCR Lab
The post-PCR lab (or dedicated area) has airborne contamination with PCR amplicon. The PCR reactions
should take place in this room. It is critical that opening of plates after PCR and handling of post-PCR
liquids is done in this room.
Reagents Required in Post-PCR Lab
Table 4.9 Reagents Required in Post-PCR Lab
Reagent Supplier Part Number
Agilent DNA1000 Kit Agilent Technologies 5067-1504
100% Ethanol Various —
NextSeqTM 500/550 Mid Output Kit v2 (150 cycles), or
NextSeqTM 500/550 High Output Kit v2 (75 cycles)
IlluminaTM FC-404-2001
FC-404-2005
PhiX Control Kit v3 IlluminaTM FC-110-3001
Nuclease-free molecular grade water Various —
1N NaOH Various —
Tween 20 Various —
DNA Binding Buffer Zymo Research D4004-1-L
DNA Wash Buffer Zymo Research D4003-2-24
TE pH 8.0 Various —
10 mM Tris HCl pH 8.3 Various —
200 mM Tris pH 7 Various —
26 Eureka™ Genotyping Workflow User Guide
Equipment Required in Post-PCR Lab
Table 4.10 Equipment Required in Post-PCR Lab
Quantity Item Supplier Part Number
1 Centrifuge with: Various —
384 plate adaptor
50 mL tube adaptor
Minimum of 1 C1000 Touch™ Thermal Cycler with 384-well
block
Bio-Rad Laboratories 1851138
1 Microcentrifuge Various —
1 Pipette, single channel (10 μL) Various —
1 Pipette, single channel (20 μL) Various —
1 Pipette, single channel (200 μL) Various —
1 Pipette, single channel (1000 μL) Various —
1 NextSeqTM 500 System IlluminaTM SY-415-1001
1 2100 Bioanalyzer Agilent Technologies G2939AA
1 Vortex mixer Various —
Eureka™ Genotyping Workflow User Guide 27
Labware Required in Post-PCR Lab
Table 4.11 Labware Required in Post-PCR Lab
Labware Supplier & Part
Number
Required for 384 Rxn
Image
384 Individual
well reservoir,
90 mL
Part of the
Eureka™
Consumables
Kit P/N
902911
1 Reservoir
2 mL collection
tubes
Zymo
Research
P/N C1001-
50
2 Tubes
Zymo-Spin™
V column
Zymo
Research
P/N C1016-
25
1 Column
28 Eureka™ Genotyping Workflow User Guide
Labware Supplier & Part
Number
Required for 384 Rxn
Image
1.5 mL LoBind
microfuge
tubes
Eppendorf
P/N
022431021
4 Tubes
50 mL
Falcon™ tubes
Corning
P/N 352070
1 Tube
10 mL
serological
pipettes
Various Multiple
Lab tape (high-
quality)
Various
Eureka™ Genotyping Workflow User Guide 29
Preparation of DNA Wash Buffer
The DNA wash buffer (Zymo Research) requires the addition of ethanol before the first use. Add 96 mL of
100% ethanol or 104 mL of 95% ethanol to the bottle containing 24 mL of DNA Wash buffer concentrate.
Cap the bottle tightly and mix. Indicate that ethanol has been added by checking the box on the label. The
DNA wash buffer is now ready for use, and may be stored at room temperature.
VIAFLO 384 Programs using a 12.5 μL Head
Prior to starting the Eureka Genotyping Assay, please verify the following programs are loaded onto the
VIAFLO 384 instrument.
EG_PROBETODNA
EG_HYBTOLIG
EG_LIGTOPCR
EG_PROBEDISP
EG_LIGDISP
EG_PCRMIXDISP
EG_SMPLEINDX
If any of the protocols are missing, contact your local Field Application Specialist (FAS).
IMPORTANT: Do not wipe down areas near electrical connections with the 2 to 5% chlorine bleach solution. Bleach damages electrical connections.
Ensure that you are using the recommended tips, 384-plates, reservoirs, and cool blocks.
The programs entered by manufacturer and verified by the FAS are adjusted to work
only with recommended reservoirs, plates, cool blocks, and tips.
The programs have also been adjusted for each individual instrument and cannot be
transferred between units without verifying the values.
Use of other tips, plates, reservoirs, or cool blocks, or transferring programs causes
inaccurate liquid transfer. The consequences range from tips bending, scraping across
plates, sample contamination, and sub optimal results.
30 Eureka™ Genotyping Workflow User Guide
Chapter 5 Fill 384 Plates with Eureka™ Genotyping Assay Reagents
Eureka™ Genotyping Assay utilizes three reagents (Eureka™ Genotyping Panel, Eureka™ Ligation
Solution, and Eureka™ PCR Master Mix) that are dispensed to all wells of their respective 384 plates before
use in the assay. The Eureka Genotyping Panel is dispensed on day 1 of the workflow. The Eureka Ligation
Solution and the Eureka PCR Master Mix are dispensed on day 2 of the workflow. The sample index plate
is already dispensed in 384 format. Sufficient volume is provided to deliver the desired volumes to each
well and account for excess needed for pipetting using the VIAFLO 384 and the 12 column reservoir.
Load Tips for Plate Filling Operations
The VIAFLO 384 requires use of the correct column of the 12 column reservoir and only two columns of
tips to dispense reagents from their source vials into the appropriate Twin.TecTM PCR Plate 384.
IMPORTANT: When loading the 32 tips, it is important to use the low power loading mode of the VIAFLO 384. The pressure of normal power mode can result in poor tip attachment to VIAFLO instrument and inaccurate dispensing.
Follow the steps below.
1. Arrange one tip box so that tips are only present in the two right most columns of the box. (There
should be exactly 32 tips.)
2. Place that tip box in position A or position B of the VIAFLO 384 instrument (see Figure 2.1 for locations
of positions A and B).
3. Align the two right most columns of the VIAFLO 384 head with the tips in the tip box.
4. Lower the pipetting head down until the tip load button on the top of the instrument flashes.
DO NOT PRESS THE TIP LOAD BUTTON YET.
5. Press the OK button in the center of the knob of side cover on the instruments control unit. This puts
the instrument in low power loading mode.
6. Now that the instrument is in low power mode, load tips normally.
7. Inspect the tips to be sure that all are securely fastened. If not, repeat the loading process.
TIP: The sound of the tips engaging with the head is different in low power mode compared to normal mode, but it is critical that the tips are fully seated. If the tips are not fully seated the pipetting height will be off and the programs will not transfer the liquids appropriately.
Eureka™ Genotyping Workflow User Guide 31
Chapter 6 Eureka™ Genotyping Assay Protocol
Figure 6.1 Eureka™ Genotyping Assay Workflow
Preparing the Genomic DNA 384 Plate
Refer to Chapter 3 for genomic DNA requirements.
This DNA plate can be made up in advance and stored at 4°C for short term storage or –20°C for longer
term storage.
At the start of the assay, dispense 3.5 μL DNA (42 ng/μL in TE, pH 8.3) per well into a green Twin.TecTM
PCR Plate 384 (four of the 384 wells should contain only 3.5 μL of the provided control DNA as the sample).
Dispense the control DNA into 4 empty wells. After addition of all the DNA, heat seal and spin the plate.
32 Eureka™ Genotyping Workflow User Guide
Stage 1: DNA Heat Fragmentation
Duration
Table 6.1 Time Required for Stage 1: DNA Fragmentation
Activity Time
Hands-on time 5 min
Fragmentation incubation 15 min
Total 20 min
Materials Required
Twin.Tec PCR Plate 384 (green)
Aluminum heat seal
Procedure
1. Spin down the sealed green plate containing gDNA.
2. Place the sealed green gDNA plate into a 384 well thermal cycler that has been programmed with the
Eureka Genotyping Assay programs.
3. Start the EG_Heat_Frag thermal cycler program.
4. After the heat fragmentation is completed, the plate is held at 25°C for no longer than 30 minutes. The
plate can be removed as soon as the thermal cycler indicates the block reaches 25°C.
TIP: The Genotyping panel can be dispensed into the blue plate while the heat fragmentation occurs.
Eureka™ Genotyping Workflow User Guide 33
Dispense the Eureka™ Genotyping Panel (Just Prior to Use)
Figure 6.2 Transfer of Eureka™ Genotyping Panel to Twin.TecTM PCR Plate 384 (blue)
Duration
The time required to dispense the Eureka Genotyping Panel is approximately 10 minutes.
Reagents and Materials Required
Eureka Genotyping Panel (2.5 mL)
12 column reservoir
Twin.Tec PCR Plate 384 (blue)
VIAFLO PCR 384 Cooling Plate
12.5 μL filtered GripTips
34 Eureka™ Genotyping Workflow User Guide
Procedure
1. Thaw and hold the Eureka Genotyping Panel at room temperature.
2. Load two columns of 12.5 μL tips on the far right side of the VIAFLO 384 head. Remove the tip box
from the VIAFLO. See Load Tips for Plate Filling Operations.
3. Re-suspend the Eureka Genotyping Panel by inverting the tube or vortexing briefly for about 5 seconds.
4. Pipet or pour the entire tube of Eureka Genotyping Panel (2.5 mL) into the last (right side) column of a
12 column reservoir.
5. In position A of the VIAFLO 384, place the 12 column reservoir with the liquid on the right side. In
position B of the VIAFLO 384, place a VIAFLO PCR 384 Cooling Plate with an empty blue Twin.Tec
PCR Plate 384 with well A1 in upper left orientation (Figure 6.3).
Figure 6.3 VIAFLO 384 layout for transfer of Eureka™ Genotyping Panel into Twin.TecTM PCR Plate 384
(blue)
NOTE: The two columns of tips on the right side both aspirate solution from the same column of the 12 column reservoir.
6. Transfer 4 μL of the Eureka Genotyping Panel to each well of the blue 384-well plate using the VIAFLO
384 program: EG_PROBEDISP.
7. Once the program has finished dispensing, remove the 12 column reservoir and eject the tips.
8. Because of the small volume of panel dispensed, We recommend that the genotyping panel be
dispensed immediately before use. After dispensing proceed immediately with Stage 2: Hybridization
with Eureka™ Genotyping Panel.
Eureka™ Genotyping Workflow User Guide 35
Stage 2: Hybridization with Eureka™ Genotyping Panel
Duration
Table 6.2 Time Required for Stage 2: Hybridization with Eureka™ Genotyping Panel
Activity Time
Hands-on time 5 min
Hybridization incubation 20 hrs
Total ~20:05 hrs
Reagents and Materials Required
Blue 384 PCR Plate containing Eureka Genotyping Panel
Green 384 PCR Plate containing Fragmented DNA
2 VIAFLO PCR 384 Cooling Plates
12.5 μL filtered GripTips
Aluminum heat seal
Procedure
1. Ready the prepared Twin.Tec PCR Plate 384 (blue) with 4 μL Eureka Genotyping Panel in every well
(see Dispense the Eureka™ Genotyping Panel (Just Prior to Use) for instructions on how to prepare this
plate). Because of the small volume of Eureka Genotyping Panel this plate should be made up
immediately before use.
2. Retrieve the Twin.Tec PCR Plate 384 containing the fragmented DNA (green) from the thermal cycler
(Stage 1: DNA Heat Fragmentation).
3. Spin down the DNA plate (green plate).
4. Load 384 of the 12.5 μL tips on the VIAFLO 384.
5. Place the green plate containing the fragmented DNA and the blue plate containing the Eureka
Genotyping panel on a VIAFLO PCR 384 Cooling Plate. Carefully remove the foil seal.
6. In position A of the VIAFLO 384, place the Twin.Tec PCR Plate 384 containing the Eureka Genotyping
Panel (blue) on the VIAFLO PCR 384 Cooling Plate. In position B of the VIAFLO 384, place the
unsealed Twin.Tec PCR Plate 384 containing fragmented DNA (green). The VIAFLO PCR 384 Cooling
Plates can be at room temperature or chilled.
7. Confirm that both plates are fully seated in the VIAFLO 384 Cooling Plates and level. If any bowing is
noted, push down on the plates until they remain fully seated and level. Confirm that both Twin.Tec
PCR Plate 384s are in the same orientation (i.e., well A1 is in the same place on both blocks).
8. Transfer 1.8 μL of Eureka Genotyping Panel from each well of the blue 384-well plate into the
corresponding well of the green DNA plate using the VIAFLO 384 and program: EG_PROBETODNA
9. Heat seal and spin the Twin.Tec PCR Plate 384 containing DNA and genotyping panel (green). This is
now the hybridization plate.
10. Inspect and make sure there are no bubbles at the bottom of the wells. If you see bubbles, spin again.
11. Place the Twin.Tec PCR Plate 384 containing DNA and genotyping panel (green) into the thermal
cycler.
12. Start the EG_Hyb thermal cycler program and allow 20 hours for hybridization and 1 minute at 54°C.
36 Eureka™ Genotyping Workflow User Guide
IMPORTANT: It is important that the hybridization plate is transferred immediately from 54°C to a pre-chilled VIAFLO 384 Cooling Plate. DO NOT allow the thermal cycler to complete the 54°C step unattended.
Figure 6.4 VIAFLO 384 Configured for Transferring the Eureka™ Genotyping Panel to
the DNA Plate
Eureka™ Genotyping Workflow User Guide 37
Dispense the Eureka™ Ligation Solution
Figure 6.5 Transfer of Eureka™ Ligation Solution to Twin.TecTM PCR Plate 384 (yellow)
Duration
The time required to dispense the Eureka Ligation Solution is approximately 15 minutes.
Reagents and Materials Required
Eureka Ligation Solution (5.5 mL)
12 column reservoir
12.5 μL filtered GripTips
Twin.Tec PCR Plate 384 (yellow)
VIAFLO PCR 384 Cooling Plate
38 Eureka™ Genotyping Workflow User Guide
Procedure
1. Thaw the vial of Eureka Ligation Solution at room temperature. After the solution is completely
thawed, use immediately or hold at room temperature for up to 30 min or on ice up to 2 hours.
2. Load only two columns of the 12.5 μL tips on the far right side of the VIAFLO 384 head. See Load Tips
for Plate Filling Operations.
3. Mix the ligation solution by inverting multiple times. DO NOT MIX BY VORTEXING.
4. Pipet or pour the entire tube of ligation solution (5.5 mL) into the last (right side) column of a 12
column reservoir. Mark this column with a lab marker.
5. In position A of the VIAFLO 384, place the 12 column reservoir with ligation solution in the far right
column. In position B of the VIAFLO 384, place an empty yellow Twin.Tec PCR Plate 384 on the
VIAFLO PCR 384 Cooling Plate (Figure 6.6).
NOTE: The two columns of tips on the right side both aspirate solution from the same column of the 12 column reservoir.
6. Dispense 12.5 μL of the ligation solution into each well of the yellow 384-well plate using the VIAFLO
384 program EG_LIGDISP.
7. Eject tips from the VIAFLO 384 head.
8. Proceed to Stage 3: Ligation, or hold the plate on ice for up to 15 minutes before proceeding.
9. Do not discard the 12 column reservoir, it will be used for dispensing the Eureka PCR Master Mix as
described in the section, Prepare PCR Sample Index Plate during amplification step.
Figure 6.6 VIAFLO 384 layout for transfer of Eureka™ Ligation Solution to Twin.TecTM PCR Plate 384
(yellow)
Eureka™ Genotyping Workflow User Guide 39
Stage 3: Ligation
Duration
Table 6.3 Time Required for Stage 3: Ligation
Activity Time
Hands-on time 10 min
Ligation incubation 15 min
Total 25 min
Reagents and Materials Required
Yellow 384 PCR Plate containing Eureka Ligation Solution
Green 384 PCR Plate containing Fragmented DNA and Genotyping Panel
2 VIAFLO PCR 384 Cooling Plates
12.5 μL filtered GripTips
Aluminum heat seal
40 Eureka™ Genotyping Workflow User Guide
Procedure
1. Pre-chill two VIAFLO PCR 384 Cooling Plates.
2. Place the prepared Twin.Tec PCR Plate 384 (yellow) with 12.5 μL Eureka Ligation Solution (see
Dispense the Eureka™ Ligation Solution for instructions on how to prepare this plate) onto a VIAFLO 384
cooling plate on ice or an ice-free cooling system until ready to proceed.
3. Load all 384 of the 12.5 μL tips on the VIAFLO 384.
4. As soon as the EG_Hyb program completes, immediately transfer the hybridization plate to the pre-
chilled VIAFLO PCR 384 Cooling Plate.
5. Carefully peel the foil seal from the hybridization plate (green).
6. In position A of the VIAFLO 384, place the unsealed hybridization plate (green) on a chilled VIAFLO
PCR 384 Cooling Plate. On position B of the VIAFLO 384, place the Twin.Tec PCR Plate 384 containing
ligation solution (yellow) on a chilled VIAFLO PCR 384 Cooling Plate.
7. Confirm that both plates are fully seated in the VIAFLO 384 Cooling Plates and level. If any bowing is
noted, push down on the plates until they remain fully seated and level. Confirm that both Twin.Tec
PCR Plate 384s are in the same orientation (i.e., well A1 is in the same place on both blocks).
8. Transfer 3.2 μL of hybridization reaction from each well of the green 384-well plate into the
corresponding wells of the yellow ligation solution plate using the VIAFLO 384 and program:
EG_HYBTOLIG After transfer, the VIAFLO 384 automatically mixes the solutions by pipetting up and
down 5 times.
9. The Twin.Tec PCR Plate 384 (yellow) now contains the genotyping panel hybridized to genomic DNA
and ligation solution.
10. Remove the yellow Twin.Tec Plate 384 containing the ligation reaction from the cooling plate, heat seal
the plate with foil.
11. Immediately, vortex the plate for 5 seconds at high speed on a vortexer with a flat head and return the
ligation reaction plate to the cooling plate for 1 minute.
12. Spin down the ligation reaction plate for 1 minute and return plate immediately to the cooling plate.
Optionally, the spin can be performed at 4°C. Allow the plate to cool for 1 minute on the VIAFLO PCR
384 Cooling Plate before and after spinning.
13. Start the thermal cycler program EG_Lig and allow the thermal cycler to come up to temperature
before inserting the ligation plate.
14. As soon as the thermal cycler block reaches 54°C, place the ligation reaction plate into the thermal
cycler and allow the ligation to proceed (54°C for 15 minutes).
15. After 15 minutes, the plate is held at 8°C until retrieved for the next stage. It is recommended that the
sample index PCR reaction be set up immediately after the ligation reaction completes.
TIP: OPTIONAL STOPPING POINT. The post-ligation plate can be stored at –20°C for up to a week.
IMPORTANT: The PCR reaction should be started within 2 hours of the ligation reaction completing.
Eureka™ Genotyping Workflow User Guide 41
Figure 6.7 VIAFLO 384 layout for transfer of hybridization reaction into the yellow ligation
solution plate.
Prepare PCR Sample Index Plate
Figure 6.8 Transfer of Eureka™ PCR Master Mix and Sample Index to Twin.TecTM PCR Plate 384 (red)
TIP: The PCR sample index plate can be prepared while the ligation reaction occurs.
42 Eureka™ Genotyping Workflow User Guide
Duration
The time required to prepare PCR Sample Index Plate is approximately 20 minutes.
Reagents and Materials Required
Eureka™ PCR Master Mix (3.5 mL)
Eureka™ Sample Index Plate
Twin.Tec PCR Plate 384 (red)
12 column reservoir (from Stage 3: Ligation)
12.5 μL filtered GripTips
2 VIAFLO PCR 384 Cooling Plates
Eureka™ Genotyping Workflow User Guide 43
Procedure
1. Thaw the Eureka Sample Index Plate and Eureka PCR Master Mix tube at room temperature.
2. Load only two columns of the 12.5 μL tips on the far right side of the VIAFLO 384 head. See Load Tips
for Plate Filling Operations.
3. Spin down the Eureka Sample Index Plate and place into a VIAFLO PCR 384 Cooling Plate. The metal
plate (cold or room temperature) aids in handling the Twin.Tec PCR Plate 384.
4. Follow the instructions in Chapter 2 of the Eureka™ Analysis Suite User Guide to enter the Sample index
plate barcode and Genotyping Panel information into the software.
5. Before using, mix the Eureka PCR Master Mix by inverting several times. DO NOT MIX BY
VORTEXING.
6. Pipet or pour the entire tube of Eureka PCR Master Mix (3.5 mL) into the last column on the unused
side of the 12 column reservoir. The mark made previously should help indicate which column has
been already used.
7. In position A of the VIAFLO 384, place the 12 column reservoir with PCR master mix in the far right
column. In position B of the VIAFLO 384, place an empty red Twin.Tec PCR Plate 384 on the VIAFLO
PCR 384 Cooling Plate (Figure 6.9).
8. Dispense 6.5 μL of the Eureka PCR Master Mix into each well of the red 384-well plate using the
VIAFLO 384 program EG_PCRMIXDISP.
9. Eject tips from the VIAFLO 384 head.
10. Leave the Twin.Tec PCR Plate 384 now containing Eureka PCR Master Mix (red) on the VIAFLO PCR
384 Cooling Plate in position B of the VIAFLO 384. Remove and discard the 12 column reservoir from
position A.
11. Load 384 of the 12.5 μL tips on the VIAFLO 384 from position A.
12. Remove the empty tip box from position A of the VIAFLO 384.
13. Carefully remove the seal from the Eureka Sample Index Plate (clear plate).
14. Load the VIAFLO PCR 384 Cooling Plate and Eureka Sample Index Plate into position A of the
VIAFLO 384 (Figure 6.10).
15. Confirm that both Twin.Tec PCR Plate 384s are in the same orientation (i.e., well A1 is in the same place
on both blocks).
16. Using the VIAFLO 384 and program: EG_SMPLEINDX, transfer 5 μL from each well of the sample
index plate (clear plate) into the corresponding wells of the red 384-well plate containing PCR master
mix.
17. The resulting Twin.Tec PCR Plate 384 (red) is the PCR sample index plate.
18. Remove and discard the clear index plate from position A. Seal this plate with your own adhesive seal
before discarding.
19. Eject tips from the VIAFLO 384 head.
20. When ready, proceed with Stage 4: PCR Amplification.
44 Eureka™ Genotyping Workflow User Guide
Figure 6.9 VIAFLO 384 layout for transfer of Eureka™ PCR Master Mix to Twin.TecTM PCR Plate 384 (red)
Eureka™ Genotyping Workflow User Guide 45
Figure 6.10 VIAFLO 384 layout for transfer of Sample Index PCR Reaction to Eureka™ Sample Index Plate
(clear)
46 Eureka™ Genotyping Workflow User Guide
Stage 4: PCR Amplification
Duration
Table 6.4 Time Required for Stage 4: PCR Amplification
Activity Time
Hands-on time 5 min
PCR incubation 1:15 hrs
Total ~1:20 hrs
Reagents and Materials Required
Eureka Sample Index Plate, thawed
Red 384 PCR Plate containing Sample Index PCR Reaction
Yellow 384 PCR Plate containing Ligation Reaction
12.5 μL filtered GripTips
2 VIAFLO PCR 384 Cooling Plates
Aluminum heat seal
Procedure
This PCR amplification step converts the ligated products into indexed libraries.
1. Pre-chill two VIAFLO PCR 384 Cooling Plates.
2. Load 384 of the 12.5 μL tips on the VIAFLO 384.
3. Put the prepared Twin.Tec PCR Plate 384 sample index plate (red) on a VIAFLO PCR 384 Cooling Plate
and place in position B of the VIAFLO 384 (see Prepare PCR Sample Index Plate for instructions on how
to prepare this plate).
4. In position A of the VIAFLO 384, place the Twin.Tec Plate 384 ligation reaction plate (yellow) on a
chilled VIAFLO PCR 384 Cooling Plate (Figure 6.11).
5. Confirm that both plates are fully seated in the VIAFLO 384 Cooling Plates and level. If any bowing is
noted, push down on the plates until they remain fully seated and level. Confirm that both Twin.Tec
PCR Plate 384s are in the same orientation (i.e., well A1 is in the same place on both blocks).
6. Using the VIAFLO 384 and program: EG_LIGTOPCR, transfer 1 μL of each ligation reaction (yellow)
into the corresponding wells of the red 384-well plate containing PCR sample indices.
7. The Twin.Tec PCR Plate 384 (red) is now the PCR ready plate and contains the ligated probes, sample
indexing PCR primers, polymerase and buffer.
8. Remove and discard the yellow Twin.Tec PCR Plate 384. Seal the plate with your own adhesive seal
before discarding.
9. Remove the Twin.Tec PCR Plate 384 containing DNA, ligated probes and PCR buffer (red) from the
cooling plate, heat seal and spin down. Inspect and make sure there are no bubbles at the bottom of the
wells. If you see bubbles, spin again.
10. Place the Twin.Tec PCR Plate 384 containing DNA, ligated probes and PCR buffer (red) into a thermal
cycler.
11. Start the EG_PCR thermal cycler program.
TIP: OPTIONAL STOPPING POINT. The post-amplification plate can be stored at 4°C overnight or –20°C for up to a week.
Eureka™ Genotyping Workflow User Guide 47
Figure 6.11 VIAFLO 384 layout for transfer of ligation reaction into PCR sample indices
plate.
48 Eureka™ Genotyping Workflow User Guide
Chapter 7 Post-PCR Library Pooling
Table 7.1 Time Required for Stages 1 and Stage 2: Library Pooling Clean-up, and Concentrating
Activity Time
Library pooling 10 min
Clean-up and concentrating 25 min
Total 35 min
Stage 1: Library Pooling
Materials Required
Red Twin.Tec PCR Plate 384 containing amplified DNA (post-PCR)
384 Individual Well Reservoir
Lab tape
Pooling the Library
1. Once PCR amplification is complete, retrieve the Twin.Tec PCR Plate 384 (red) from the thermal cycler.
This Twin.Tec PCR Plate 384 containing amplified DNA (red) is now post-PCR and must not be opened
except in a Post-PCR space.
2. Spin down the plate and carefully remove the foil seal from the Twin.Tec PCR Plate 384 (red). Avoid
splashing by holding the post-PCR plate tightly.
3. Tape the 384 Individual Well Reservoir upside down on top of the post-PCR plate. Make sure that all
the wells of the plate fit within the collection area of the reservoir. Use four pieces of tape, taping all
four sides.
NOTE:
Make sure that the Twin.Tec PCR Plate 384 containing PCR amplicons (red) is firmly
secured to the reservoir in the orientation to collect the liquid from the plate into the
reservoir (Figure 7.1).
Make sure the reservoir and the plate are well secured with lab tape.
Eureka™ Genotyping Workflow User Guide 49
Figure 7.1 Reservoir Taped on Top of Post-PCR Plate
4. Invert the post-PCR plate so that the reservoir is now on the bottom (Figure 7.2).
Figure 7.2 Inverted Plate After Centrifugation
5. Spin the inverted plate at 800 xg (approximately 2000 rpm) for 1 minute.
6. After the 1 minute spin time, all the liquid should be in the reservoir.
7. Remove the tape holding the reservoir and the Twin.Tec PCR Plate 384 containing DNA amplicons (red)
together.
8. Check to make sure that all of the wells of the Twin.Tec PCR Plate 384 containing DNA amplicons (red)
are empty, and then discard the Twin.Tec PCR Plate 384 containing DNA amplicons (red).
9. If any wells still contain liquid, use a pipette to remove the liquid and combine with liquid already in
reservoir.
DNA Wash Buffer
The DNA wash buffer requires the addition of ethanol before the first use. Please see Preparation of DNA
Wash Buffer for instructions.
50 Eureka™ Genotyping Workflow User Guide
Stage 2: Pooled Library Clean-up and Concentration
Reagents and Materials Required
Pooled-PCR product in the 384 individual well reservoir (from Stage 1)
DNA Binding Buffer
DNA Wash Buffer (see Preparation of DNA Wash Buffer)
TE, pH 8.0 (150 μL)
Zymo-Spin™ V column
50 mL conical tube
2 mL collection tube
1.5 mL LoBind microfuge tube
Serological pipette (10 mL)
Alcohol resistant pen
1. Tighten the bottom portion of the Zymo-Spin V column. With an alcohol resistant pen, label the Zymo-
Spin V column and place it in a 50 mL conical tube.
2. Add 10 mL of the DNA Binding Buffer to the pooled PCR product in the reservoir.
WARNING: Carefully add the DNA binding buffer to avoid accidentally splashing out the pooled library.
3. Mix the two solutions in the reservoir by slowly drawing the liquid in and out of a 10 mL serological
pipette several times. Gently rock the basin by hand to mix as well. The pooled PCR products and the
DNA Binding Buffer must be completely mixed.
4. Pipet all of the pooled PCR product and DNA Binding Buffer mixture from the reservoir into the
labeled Zymo-Spin V column that has been placed into a 50 mL conical tube.
5. Spin for 90 seconds at 2500 rpm (~500 xg) in a centrifuge with 50 mL tube adaptors. No caps needed.
6. Discard the flow-through.
7. If any liquid remains in the column, spin for additional 90 seconds at 2500 rpm (~500 xg) in a centrifuge
with 50 mL tube adaptors.
8. Discard the flow-through.
9. Pipet 5 mL of DNA Wash Buffer into the column. Be sure that ethanol has been added.
10. Spin for 90 seconds at 2500 rpm (~500 xg).
11. Discard the flow-through.
12. Pipet 5 mL of DNA Wash Buffer into the column.
13. Spin for 90 seconds at 2500 rpm (~500 xg).
14. Discard the flow-through.
TIP: If the bottom portion of the Zymo-Spin V column comes loose during centrifugation, reattach the bottom portion and pass the liquid through the column again.
15. Remove the smaller bottom portion of the Zymo-Spin V column and transfer the bottom portion to a
clean 2 mL collection tube.
16. Discard the top portion of the column and the 50 mL conical tube.
17. Add 600 μL of DNA wash buffer to the bottom portion of the Zymo-Spin V column that was placed in
Eureka™ Genotyping Workflow User Guide 51
the 2 mL collection tube in Step 15.
18. Spin at 14000 rpm (full speed, ~10,000 xg) for 1 minute in a benchtop microfuge.
19. Discard the flow-through.
20. Add 600 μL of DNA wash buffer to the column.
21. Spin at 14000 rpm (full speed, ~10,000 xg) for 1 minute.
22. Discard the flow-through.
23. Transfer the column to a new 2 mL collection tube and spin at 14000 rpm (full speed, ~10,000 xg) for 5
minutes. This is a dry step, do not add any buffer at this step.
24. Transfer the column to a labeled 1.5 mL LoBind microfuge tube.
25. Add 150 μL of TE, pH 8.0 to the column. DO NOT use Zymo Research Elution Buffer. Incubate at room
temperature for 1 minute to allow the buffer to saturate the column.
26. Spin for 1 minute at 14000 rpm (full speed, ~10,000 xg).
Save the eluate (≈140 μL) and discard the column.
27. The eluate from Step 26 is the pooled library. It can be stored at 4°C short term and –20°C long term.
This is a PCR amplified product and must not contaminate the pre-PCR clean room.
TIP: OPTIONAL STOPPING POINT. The pooled library can now be stored at –20°C for up to 2 weeks.
52 Eureka™ Genotyping Workflow User Guide
Stage 3: Library Qualification and Quantification via Agilent 2100 Bioanalyzer
Reagents and Materials Required
Pooled library (5 μL)
10 mM Tris-HCl pH 8.5 (20 μL)
LoBind 1.5 mL microcentrifuge tube
Table 7.2 Time Required for Stage 3: Library Qualification and Quantification
Activity Time
Hands-on time 15 min
Bioanalyzer runtime 45 min
Total 1 hour
It is critical that the quality of the pooled library be assessed and that the library size and quantity
be determined to calculate nanomolarity prior to blending the pooled libraries.
We recommend the following procedure, especially for determining pooled library quality.
Quantitation without considering library size distribution will compromise results.
1. In a fresh DNA LoBind 1.5 mL microcentrifuge tube, make a 1:5 dilution using 5 μL of the pooled
library with 20 μL of 10 mM Tris-HCl pH 8.5 to make the diluted library stock.
IMPORTANT: Do not use the entire pooled library to make the diluted library stock.
2. Quantify the amount of DNA in the 1:5 diluted library stock with the DNA1000 assay. Follow the
manufacturer’s protocol for the Agilent Bioanalyzer DNA1000 Kit. The Bioanalyzer assay also provides
size and quality information about the pooled library.
3. We recommend loading two wells per diluted library. Two wells are recommended in case there is an
error in one of the wells (such as an air bubble spike).
4. Figure 7.3 shows the main library peak and an example of the larger molecular weight smear.
5. Use the Bioanalyzer to calculate the nM concentration of the library peak.
6. Record this concentration for use in the Eureka Genotyping Calculator Software (refer to the Eureka™
Analysis Suite User Guide, P/N 703380).
TIP: OPTIONAL STOPPING POINT. The quantified library can be stored at –20°C longer term.
Eureka™ Genotyping Workflow User Guide 53
Figure 7.3 Representative Bioanalyzer Trace
54 Eureka™ Genotyping Workflow User Guide
Stage 4: Combining Pooled Libraries
Library Blending and Balancing
When two or more libraries are blended together for a single sequencing run, it is critical that the portion of
the blend assigned to each library is scaled to the SNP-plex of the genotyping panel of the library and the
number of sequences required. Refer to the section on Setting Up Your Sequencing Run Calculations in
Chapter 2 of the Eureka™ Analysis Suite User Guide. Combine the indicated volume of each diluted library to
create the lane blend. The sum of the volumes in the blend MUST be subtracted from the “Volume of
sequencing reaction (35- 50 μL)” entered in Run Setup window tab in the Eureka Analysis Suite. This
difference is made up with 10 mM Tris-HCl, pH 8.5 (EB Buffer) in order to achieve a final Sequencing Tube
(lane blend) concentration of 10 nM. It is recommended that the lane blend is diluted to 10 nM.
WARNING: The entries in the column “% of Blend Used (Total: 0)” from the Run Setup window tab in Eureka Analysis Suite, MUST add up to 100%.
TIP: OPTIONAL STOPPING POINT. The library blend can be stored at –20°C longer term.
Eureka™ Genotyping Workflow User Guide 55
Chapter 8 Sequence Data Generation
1. Be sure to complete Stage 3: Library Qualification and Quantification via Agilent 2100 Bioanalyzer and Stage
4: Combining Pooled Libraries before proceeding.
2. Whether a mid output or a high output flow cell is used depends on the number of samples and the
Eureka Genotyping Panel. The calculator in the Eureka™ Analysis Suite assists in this determination.
3. Make 0.2N NaOH according to the IlluminaTM NextSeq500 System User Guide (800 μL molecular grade
water + 200 μL 1N NaOH). This must be made fresh for every run.
4. Transfer 9.5 μL of the 10 nM library blend, created in Library Blending and Balancing, to a new 1.5 mL
microcentrifuge tube.
5. Add 0.5 μL of the 10 nM phiX (IlluminaTM, cat# FC-110-3002) to the 9.5 μL 10 nM library blend from
Step 4.
6. Add 15 μL of 10 mM Tris-HCl, pH 8.5 to the 10 μL library blend + phiX mix from Step 5. This is now
the 4 nM lane + phiX mix.
7. Vortex 5 seconds, quick spin down.
8. Transfer 5 μL of the 4 nM mix from Step 6 to a new microcentrifuge tube.
9. Add 5 μL of the freshly made 0.2N NaOH to the 5 μL of 4 nM mix from Step 8.
10. Vortex 5 seconds, quick spin down.
11. Incubate at room temperature 5 minutes.
12. After 5 minutes, add 5 μL of 200 mM Tris pH 7.
13. Vortex 5 seconds, quick spin down.
14. Add 985 μL of cold HT1 buffer (Illumina supplied). This is now the 20 pM denatured lane blend.
15. Vortex 5 seconds, quick spin down.
16. Make a 1.4 pM dilution for loading onto flow cell using the 20 pM denatured lane blend and HT1
buffer according the IlluminaTM NextSeq500 System User Guide. (Based on the density of the first run, the
initial 1.4 pM loading amount should be increased/decreased to optimize cluster density.)
17. Ensure that the sequence data being generated is saved into a destination where it can be accessible by
the Eureka Analysis Suite software.
18. Proceed with NextSeq run setup. In the Run Setup page, Read1 should be entered as 40 and Index 1
should be entered as 15 as shown in Figure 8.1.
TIP: Read1 and Index1 are required.
WARNING: The Eureka Analysis Suite software will crash if Read2 is selected.
56 Eureka™ Genotyping Workflow User Guide
Figure 8.1 NextSeq 500 Run Settings
Eureka™ Genotyping Workflow User Guide 57
Chapter 9 Processing Multiple Plates per Week in the Eureka™ Genotyping Workflow
Overview of Eureka™ Genotyping Workflows
Using the Eureka™ Genotyping Solution there are many workflows to process multiple 384 plates in a 5
day work week. This chapter presents the timing of the steps required to process either 8 or 32 plates in five
days. The 8-plate workflow requires only a headcount of one, while the 32-plate workflow requires a
headcount of two. If processing 8 plates weekly, this is 400 plates of 380 samples + 4 control DNA for a total
of 153,600 samples in 50 weeks. If processing 32 plates weekly, this is 1600 plates of 380 samples + 4 control
DNA for a total of 614,400 samples in 50 weeks.
IMPORTANT: Experienced users and careful timing are critical for the successful execution of this workflow.
Assumptions
The information presented in this chapter is based on several assumptions:
8-plate Workflow
Assumes user familiarity with all procedures required for the Eureka™ Genotyping Assay as
described in this user guide.
Head count of one
One INTEGRA VIAFLO 384 Instrument
Thermal cyclers = 2 (384)
Cooling blocks = 2 (384)
5 day work week, 9 AM—5 PM
Source sample DNA
32-plate Workflow
Assumes user familiarity with all procedures required for the Eureka Genotyping Assay as
described in this user guide.
Head count of two
One INTEGRA VIAFLO 384 Instrument
Thermal cyclers = 8 (384)
Cooling blocks = 8 (384)
5 day work week, 9 AM—5 PM
Source sample DNA
58 Eureka™ Genotyping Workflow User Guide
Figure 9.1 shows the workflow to create pooled libraries from 8 x 384 DNA plates in one week, and
Figure 9.2 shows the workflow to create pooled libraries from 32 x 384 DNA plates in one week.
Figure 9.1 Eureka™ Genotyping Assay 8-plate Workflow (8 x 384)
Figure 9.2 Eureka™ Genotyping Assay 32-plate Workflow (32 x 384)
Eureka™ Genotyping Workflow User Guide 59
Appendix A Eureka™ Genotyping Workflow Assay FAQs and Troubleshooting
FAQs
Q: What should I do if I have less than 384 samples?
A: The kit is meant for 384 reactions. It cannot be divided into smaller subsets. Run the assay with 384
samples. If you must run with fewer samples, instead of DNA put 500 mM EDTA pH 8.0 into the
empty wells of the 384-well plate and proceed with the workflow.
Q: I did not fragment the gDNA before adding the genotyping panel to it, can I still proceed?
A: Although not recommended, heat fragment the DNA with the genotyping panel and proceed.
Q: The supplied control DNA was not used. Can I proceed?
A: The control DNA is used by Thermo Fisher Scientific for troubleshooting. It is used also to track the
orientation of the sample plates.
Q: How many freeze/thaw cycles can the Eureka™ Genotyping Panel undergo?
A: The Eureka Genotyping Panel can undergo one freeze/thaw cycle.
Q: How many freeze/thaw cycles can the Eureka™ Ligation Solution undergo?
A: The Eureka Ligation Solution can undergo one freeze/thaw cycle.
Q: How many freeze/thaw cycles can the Eureka™ PCR Master Mix undergo?
A: The Eureka PCR Master Mix can undergo one freeze/thaw cycle.
Q: How many freeze/thaw cycles can the Eureka™ sample index plate(s) undergo?
A: The Eureka sample index plate(s) can undergo one freeze/thaw cycle.
Q: During the PCR amplification step, the sample index plates were accidentally switched. What do I do?
A: Assign the correct index to each sample using Eureka Genotyping Calculator.
Q: Why do two pooled libraries have the same sample indices assigned?
A: Both plates were accidentally PCR amplified using the same sample index plate. Ensure these two
libraries are not combined in the same sequencing lane. Generate data using different sequencing
runs.
Q: The 384-well plate was accidentally dropped after the PCR amplification step was complete. How can
I proceed forward?
A: If the plate is still sealed, spin it down to collect the contents at the bottom of each well and proceed
with the next step of the assay. If the plate is not sealed, repeat the PCR amplification step from the
post-ligation reaction using a fresh sample index plate and PCR master mix. Decontaminate the area
where the plate dropped. These amplicons can cause cross-contamination for future reactions.
60 Eureka™ Genotyping Workflow User Guide
Troubleshooting
Table A.1 Eureka™ Genotyping Workflow Assay Troubleshooting
Observation Possible Cause Solution
Post-hybridization wells appear
dry.
Poor sealing of the wells. Re-test the heated plate sealer settings and
ensure that the plate is sealed properly.
Possible VIAFLO pipetting
issue during transfer of
reagents into 384- well
plate.
Observe liquid volume in INTEGRA tips
during transfer steps and check volumes in the
wells after transfer. If issue persists contact
INTEGRA support.
The thermal cycler lid
failed, causing
condensation at the top of
the plate seal.
Ensure that the heated lid of the thermal cycler
is working properly.
The sealed plate was
dropped or shaken,
disturbing the contents of
the plate.
Spin down the 384-well plate to collect the
contents of at the bottom of each well, and
then proceed with the assay.
Post-hybridization the wells
have variable volumes.
Poor sealing of the wells. Re-test the heated plate sealer settings and
ensure that the plate is sealed properly.
Possible VIAFLO pipetting
issue during transfer of
reagents into 384- well
plate.
Observe liquid volume in INTEGRA tips
during transfer steps and check volumes in the
wells after transfer. If issue persists contact
INTEGRA support.
The thermal cycler lid
failed, causing
condensation at the top of
the plate seal.
Ensure that the heated lid of the thermal cycler
is working properly.
The sealed plate was
dropped or shaken,
disturbing the contents of
the plate.
Spin down the 384-well plate to collect the
contents of at the bottom of each well, and
then proceed with the assay.
Assay was started with
variable input amounts of
gDNA.
Check DNA volume into the reaction. Proceed
- it could work.
Pipette tips on the INTEGRA
VIAFLO 384 Instrument are at
the edge of the wells of the 384-
well plate.
The plate is not fully seated
on the VIAFLO 384 cooling
block.
Ensure that the 384-well plate is firmly seated
on the VIAFLO PCR 384 Cooling Plate and
proceed with the assay.
Eureka™ Genotyping Workflow User Guide 61
Observation Possible Cause Solution
The VIALFO 384 alignment
is off.
Power cycle the VIAFLO 384 Instrument and
proceed with the assay. If this does not resolve
the issue, call INTEGRA Support.
The well openings of the
384-well plate are smaller
due to overheating caused
during sealing of the plate.
Re-test the heated plate sealer settings and
ensure that the plate is sealed properly and
that the wells are not overheated.
The 384 head is not seated
properly in the VIAFLO
384 Instrument.
Make sure the 384 head is well seated into the
VIAFLO 384 Instrument, or call INTEGRA
Support if you need further assistance.
Pipette tips on the INTEGRA
VIAFLO hit the sides or bottom
of the 384-well plate.
VIAFLO 384 Instrument
maybe misaligned due to
incorrect settings, causing
the pipette tips to hit the
sides of the 384-well.
Call INTEGRA Support to re-calibrate the
instrument.
Storing the VIAFLO PCR
384 Cooling Plate at –20°C
may cause condensation to
freeze inside of the
VIAFLO PCR 384 Cooling
Plate, causing the pipette
tips to hit the bottom of the
384-well plate.
Do not store the VIAFLO PCR 384 Cooling
Plate at –20°C.
The 384-well plate might be
slightly bent or warped
due to the thermal cycler
incubation.
If this occurs at one of the optional stopping
points during the assay for e.g., Postligation,
freeze the reactions. Once the instrument issue
is resolved, thaw the reactions and proceed
with the assay. If this is not an optional
stopping point in the assay, start again.
The tips are not seated
properly in the mandrels of
the 384-well head.
Ensure that the plate is well seated in the
VIAFLO PCR 384 Cooling Plate. If you see
bowing or warping of the plate, press down on
the four corners.
PCR amplicons spill during
addition of binding buffer
during the pooled library clean
up and concentration step.
Forceful addition of the
DNA binding buffer to the
PCR amplicons in the
collection reservoir.
Redo the PCR amplification step from the
post-ligation reaction. Gently add the DNA
binding buffer to the PCR product during the
pooled library clean up and concentration step.
Decontaminate area where the PCR amplicons
split to avoid future cross-contamination. No Bioanalyzer peaks observed
for pooled libraries during
library QC step.
No PCR master mix was
added to the samples prior
to PCR step.
Repeat the PCR amplification step ensuring
use of PCR master mix along with the sample
indices.
62 Eureka™ Genotyping Workflow User Guide
Observation Possible Cause Solution
No genotyping panel
added to the gDNA
samples prior to
hybridization.
Repeat assay ensuring addition of genotyping
panel prior to overnight hybridization step.
Samples were in correctly
diluted prior to loading the
Bioanalyzer chip.
Ensure proper dilution of the pooled libraries.
Samples were lost during
the purification steps due
to handling errors.
Repeat the PCR and purification carefully
following the protocol.
The Bioanalyzer trace does not
have the high molecular weight
smear that is shown in the user
guide.
This high molecular weight
smear can be caused by
over amplification. If the
pooled library is run on a
denaturing gel the high
MW smear often
disappears.
The smear is not required. It is only sometimes
present. If there is the single 220 bp peak
present the library can proceed to sequencing.
The Bioanalyzer peak is at 210
bp and not 220 bp.
The Bioanalyzer peaks may
vary due the content of the
genotyping panel
If your Bioanalyzer peak is between 210 and
230 bp (commonly at 220 bp) proceed with the
sequencing run
The Bioanalyzer trace has a peak
at 220 bp and other secondary
peaks.
Multiple secondary peaks
may be caused due to
insufficient ligation
product produced.
If the peak at 220bp is 2X the nM of the other peak, the pooled library can be sequenced. As the Bioanalyzer quantification includes both peaks the realized read depth will likely be different than estimated.
If the other peak is equivalent or larger nM the library should not be sequenced and the assay must start from the beginning.
The Bioanalyzer trace peak is at
150 bp and not 220 bp.
Bioanalyzer ladder has
shifted.
If there are 13 peaks in ladder well and the
Bioanalyzer failed to recognize all 13,
manually include the missing peak. If there are
not 13 peaks in the ladder, rerun the
Bioanalyzer QC on a fresh chip. If the
Bioanalyzer adjustments resolve the issue the
pooled library can be sequenced.
Eureka™ Genotyping Workflow User Guide 63
Observation Possible Cause Solution
Insufficient DNA
concentration or quality in
the original samples.
If there is a low amount of input DNA in the
original samples, the assay will produce noise
that can appear as a 150 bp peak. The amount
of starting DNA needs to be increased. The
pooled library should not be sequenced. The
assay must start from the beginning with
higher concentration/ quality of DNA used.
Assay workflow issue
resulting in hybridization
or ligation failure.
If the hybridization or ligation failed a small
MW band appears due to PCR artifacts. The
pooled library should not be sequenced. The
assay must start from beginning.
No ligation solution was
added to the samples.
Repeat the assay ensuring the addition of the
ligation solution.
The sequencer produced fewer
reads than expected.
Cluster density during
loading of the sequencer
was not optimized.
The sequencer was validated with Illumina
and phiX from Illumina. Based on the cluster
density of a recent run or the phiX validation
run, increase or decrease the lane blend
amount from the 1.4 pM dilution
recommended in the user guide. The pooled library was
quantified incorrectly.
See Bioanalyzer related observations above.
phiX alignment percentage in
the sequencing run summary is
zero.
phiX sample is degraded. Contact Illumina support.
phiX sample was not
loaded onto the sequencer.
Load the phiX sample during the next
sequencing run.
Failed samples. Low DNA quantity
(recommended 42ng/jL) or
quality
Use the recommended DNA concentration and
rerun the assay.
Improper sealing of the
384-well plate.
Determine if there is a pattern to the failed
samples as seen in the plate view of the
Eureka™ Analysis Suite software. If the
failures are along the edge it suggests an issue
with the sealing or with the thermal cycler. Re-
test the heated plate sealer and ensure the
thermal cycler is functioning. Reagent transfer issues
during the assay.
Observe the volume of liquid transferred
during the various steps of the assay by the
VIAFLO 384 Instrument. Call INTEGRA
Support if needed. Varying genetic
background of the sample.
Reset threshold settings. Contact Support for
help with resetting thresholds if needed.
Sample wells were
incorrectly labeled as
empty.
Correct the plate layout and rerun Eureka
Analysis Suite software.
Incorrect threshold setting
are used.
Correct the threshold settings and rerun
Eureka Analysis Suite software.
64 Eureka™ Genotyping Workflow User Guide
Observation Possible Cause Solution
No calls for samples along the
edge of a 384-well plate.
The post-PCR plate (red) is
not seated properly on the
collection reservoir during
centrifugation.
Repeat the PCR amplification from the post-
ligation reaction. Ensure that the post-PCR
plate is secured well to the collection reservoir
during centrifugation.
Poor sealing at the edge of
the 384- well plate.
Re-test the heated plate sealer and adjust
settings as needed to ensure proper sealing of
the 384-well plate.
Summary Tab in the Viewer
displays all samples failed.
Incorrect panel name and sample index plate combination entered in Eureka Analysis Suite software
Eureka Analysis Suite software not pointed to correct sequence data folder.
Check the Summary Tab in Eureka Analysis
Suite. Contact Support for assistance.
There are no calls for the control
DNA.
Control DNA was not
loaded at the beginning of
the assay.
Ensure that control DNA is added to the 384-
well plate at the beginning of the assay.
A reagent was omitted
during the assay.
Repeat the assay ensuring the proper reagent
is added at every step.
More AB genotypes than
expected are being called.
Inbred penalty file was not
used during analysis.
Re-run Eureka Analysis Suite software using
an inbred penalty file. An inbred penalty value
of 16 should be used on all samples that are
inbred lines. If this is not known, give all
samples a low penalty of 4, and examine the
results to see if too many heterozygotes are
still being called and a higher penalty is
needed. Possible cross-
contamination of samples.
Each well should be an individual sample.
Rerun assay with individual samples. Check
for and eliminate sources/causes of
contamination of samples. Loci previously in Poly High
Res (PHR) category are no
longer PHR.
Samples have lower than
DNA concentration.
Check and adjust the DNA concentration and
quality as needed.
Samples with varied
genetic content.
Due to genetic drift of a different genetic
population proximal SNPs can change the SNP
category for a locus. The SNP category can
change if the panel is used on samples that
have a different genetic context than the
population the panel was designed for. Different number of
samples processed.
The generation of the genotype space for any
locus is based on the samples. If SSP have not
been generated contact your genotyping
specialist to discuss SSP.
Eureka™ Genotyping Workflow User Guide 65
Observation Possible Cause Solution
Incorrect locus start and
locus length used in Eureka
Analysis Suite software.
Ensure that the locus start is 6 and the locus
length is 12.
Used generic priors instead
of SNP Specific priors file.
Rerun Eureka Analysis Suite software with
correct priors file.
The genotypes of the samples
seem to be mixed up/ the
samples have unlikely
genotypes.
Sample sheet layout is not
correct.
Check the sample sheet layout and re run
Eureka Analysis Suite software with corrected
sheet.
A plate was rotated 180 at
some step of the workflow.
If you are sure this has occurred adjust the
sample sheet and rerun Eureka Analysis Suite
software.
A few of the loci have no
genotypes.
Genetic background of the
samples is not the same as
the samples used to
develop the custom panel.
The assay was custom designed for a specific
genetic background. If this context has
changed samples may not produce genotypes
on some loci.
There are four clusters instead of
the expected three.
Variable genetic
background has created
more clusters.
Visually examine the cluster plots to determine
how to proceed with the generated genotypes.
Contact your specialist for assistance.
66 Eureka™ Genotyping Workflow User Guide
Documentation and support
Obtaining support
Technical support For the latest services and support information for all locations, visit
www.thermofisher.com.
At the website, you can:
Access worldwide telephone and fax numbers to contact Technical Support
and Sales facilities
Search through frequently asked questions (FAQs)
Submit a question directly to Technical Support (thermofisher.com/support)
Search for user documents, SDSs, vector maps and sequences, application
notes, formulations, handbooks, certificates of analysis, citations, and other
product support documents
Obtain information about customer training
Download software updates and patches
Safety Data Sheets (SDS)
Safety Data Sheets (SDSs) are available at thermofisher.com/support.
Limited product warranty
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thermofisher.com
23 January 2017